弓形虫真核表达质粒pcDNA3/wx2的构建与鉴定

Construction and identification of the recombinant plasmid pcDNA3/wx2 from Toxoplasma gondii

目的构建弓形虫新基因wx2的真核表达质粒,以其做为弓形虫DNA疫苗研究其保护性。方法PCR扩增出编码新基因wx2ORF,用EcoRI/XhoⅠ分别对扩增产物和pcDNA3进行双酶切,将wx2ORF定向克隆到pcDNA3的EcoRI/XhoⅠ位点,对重组质粒进行PCR,双酶切初步鉴定后做序列测定。结果特异扩增出预期的wx2ORF片段,大小为570bp左右,扩增产物经双酶切后成功连接到pcDNA3中,经PCR,双酶切及序列测定表明重组质粒中含有wx2读框。结论成功构建弓形虫真核表达质粒pcDNA3/wx2。

The purpose of this study is to construct a vaccine vectors the new gene wx2 of Toxoplasma gondii in order to provide the foundation for exploring the protective immune efficacy. The opening reading frame（ORF） of the wx2 gene was amplified by PCR. The PCR product and plasmid pcDNA3 were digested with EcoR Ⅰ and XhoⅠ respectively, then ligated into the pcDNA3 at polylinker. The recombinant vector pcDNA3/wx2 was characterized by PCR, restriction enzyme digestion and sequencing analysis. The sequence results shown that the recombination plasmid pcDNA3/wx2 contained wx2 ORF with the right orientation,and the eukaryotic expression vector pcDNA3/wg2 is successfully constructed.